Underlying the human erythrocyte plasma membrane is a filament network that consists of short actin filaments cross-linked into a regular hexagonal lattice by long spectrin molecules in association with a helper protein, 4.1. The long range goal of this research is to determine how this membrane skeleton is assembled during erythroid differentiation and how its molecular organization is functionally related to cell shape and membrane deformability. Recently, I have identified a novel, non-actin Mr 42,000 tropomyosin-binding protein in the membrane skeleton. The Mr 42,000 protein is present in the same number of copies as the number of actin filaments in the membrane skeleton (30,000/cell) and the erythrocyte tropomyosin is sufficiently abundant for two molecules to be associated with each short actin filament. The possibility that these proteins are associated with the actin filaments in situ in the membrane skeleton will be examined morphologically by immuno-gold labelling techniques in negatively stained membrane skeletons and biochemically by measuring radiolabelled tropomyosin binding to membrane skeletons depleted of tropomyosin. The role of the Mr 42,000 protein in modulating tropomyosin association with the membrane skeleton will be evaluated by competition-binding experiments in the presence of soluble Mr 42,000 protein and by measuring its effect on tropomyosin binding to purified F-actin. The possibility that these proteins, or another component extracted from membranes, could cap the end(s) of the short actin filaments will be examined by testing their ability to block actin filament elongation from a spectrin-protein 4.1-actin complex, using a non-perturbing fluorometric assay to monitor actin polymerization. Reticulocytes isolated from the circulation of anemic rats will be used as a model system to initiate an exploration of the role of the membrane skeleton in the biogenesis of the biconcave shape characteristic of mature erythrocytes. It is planned to characterize the biosynthesis and incorporation of actin in the membrane skeleton, to determine the proportions of cytoplasmic and membrane-associated actin as monomer and filament, to determine the lengths of actin filaments in the membrane skeleton and whether their ends are capped, and to study the ultrastructural organization and localization of spectrin, actin, tropomyosin and other components in negatively stained membrane skeletons.